The spatial control of cytokinesis is an important aspect of development in multicellular organisms. Division plane positioning is not only critical for the proper segregation of chromosomes and organelles to daughter cells, but can also influence the developmental fates of the daughter cells. The spatial control of cytokinesis is of particular significance in plant development, where cells are embedded in a rigid extracellular matrix, the cell wall: the relative positions of cells within plant tissues are fixed at birth by planes of cell division. Although the processes of division plane determination and cytokinesis are in some respects different in animals and plants, in both cases they involve cytoskeleton-mediated interactions between the nucleus/microtubule organizing centers and the cell cortex that position the cytokinetic apparatus appropriately within the dividing cell. The molecular mechanisms underlying these interactions are poorly understood in both animals and plants, and are currently being studied by means of biochemical and genetic methods. The project described in this proposal represents a genetic approach to understanding the spatial control of cytokinesis in a higher plant, Zea mays. Because of the lack of relative cell movement in plant tissues, mutants with alterations in cell division pattern can be recognized by means of the resulting alterations in the regular cell pattern of the mature maize leaf. The proposal concentrates on the analysis of a gene required for the spatial control of cytokinesis that was identified in this way, Tangled (Tan). More specifically, the phenotype of this mutant indicates that the Tan gene is required for elongated cells to divide in a plane parallel to the cell's long axis. Specific Aim l is to develop a better understanding of the function of this gene by investigating the cellular basis of the tan mutant defect. This will be accomplished by means of comparisons between mutant and wild-type leaves with respect to: 1) cytoskeletal rearrangements m dividing cells, 2) aspects of cell division that can be observed in living tissues by video microscopy, and 3) divisions in various tissues throughout the plant involving cells of different shapes. The tan mutation was caused by the insertion of a transposable element; fragments of the Tan gene adjacent to the transposon insertion site have already been cloned. Specific Aim 2 is to complete the cloning of the Tan gene, analyze the predicted amino acid sequence of its product for homology to other proteins, and examine the tissue and subcellular localization of this protein to try to understand at a molecular level its involvement in spatial aspects of cytokinesis. Specific Aim 3 is to use genetic mosaic analysis to determine whether the Tan gene functions cell autonomously, and to explore the role of cell-cell interactions in the control of cell division pattern in plant tissues. Finally (Specific Aim 4), further screening for recessive mutations altering the cell pattern of the maize leaf will be done to identify other genes required for the spatial control of cell division in plants. Insights gained from these studies will contribute to our understanding of mechanisms regulating cell division in eukaryotic organisms that are essential for normal development, and the loss of which is associated with neoplastic diseases in humans and animals.